Recombinant Human Placental Growth Factor Research Articles

PlGF/FLT-1 deficiency leads to reduced STAT3-C/EBPβ signaling and aberrant polarization in decidual macrophages during early spontaneous abortion.

Dysregulated macrophage polarization (excessive M1-like or limited M2-like macrophages) in the early decidua contributes to allogeneic fetal rejection and thus early spontaneous abortion. However, the modulators of M1/M2 balance at the early maternal-fetal interface remain mostly unknown. First-trimester decidual tissues were collected from normal pregnant women undergoing elective pregnancy terminations and patients with spontaneous abortion. We measured the expression of placental growth factor (PlGF) and Fms-like-tyrosine-kinase receptor 1 (FLT-1), and characterized the profiles of macrophages in decidua. Notably, we investigated the effect of recombinant human PlGF (rhPlGF) on decidual macrophages (dMφs) from normal pregnancy and revealed the underlying mechanisms both in vitro and in vivo. The downregulated expression of PlGF/ FLT-1 may result in spontaneous abortion by inducing the M1-like deviation of macrophages in human early decidua. Moreover, the CBA/J×DBA/2 abortion-prone mice displayed a lower FLT-1 expression in uterine macrophages than did CBA/J×BALB/c control pregnant mice. In in vitro models, rhPlGF treatment was found to drive the M2-like polarization of dMφs via the STAT3/CEBPB signaling pathway. These findings were further supported by a higher embryo resorption rate and uterine macrophage dysfunction in Pgf knockout mice, in addition to the reduced STAT3 transcription and C/EBPβ expression in uterine macrophages. PlGF plays a key role in early pregnancy maintenance by skewing dMφs toward an M2-like phenotype via the FLT-1-STAT3-C/EBPβ signaling pathway. Excitingly, our results highlight a rationale that PlGF is a promising target to prevent early spontaneous abortion.

Cardiovascular and hemodynamic consequences of recombinant placental growth factor administration in Guinea pigs

ABSTRACT Placental growth factor (PlGF), a member of the vascular endothelial growth factor family of proteins regulating angiogenesis, has been shown to have acute vasodilatory effects on human resistance arteries. However, the acute hemodynamic effects of PlGF in vivo are not known. The aim of this study was to report acute cardiovascular changes induced by recombinant human PlGF administered intravenously in male Guinea Pigs with implanted telemeters. PlGF decreased mean arterial blood pressure by 10–20% within minutes. The magnitude of reduction was similar at three dose levels; however, the duration of relative hypotension was dose-dependent. Blood pressure reduction resulted in a compensatory increase in heart rate, or reflex tachycardia. To rule out any direct effect on the heart, PlGF was tested in the ex vivo Langendorff heart preparation, and no cardiac changes were found. Together these results suggest that the PlGF-related changes in blood pressure are largely mediated by its actions in the vasculature.

A literature review and best practice advice for second and third trimester risk stratification, monitoring, and management of pre‐eclampsia

A literature review and best practice advice for second and third trimester risk stratification, monitoring, and management of pre‐eclampsia

Administration of recombinant human placental growth factor decreases blood pressure in obese hypertensive pregnant rats.

Although epidemiological studies have shown that obesity is associated with increased incidence of hypertension during pregnancy, the mechanisms linking these two comorbidities are not as well studied. Previous investigations detected lower levels of the anti-hypertensive and pregnancy-related factor, placental growth factor (PlGF), in obese hypertensive pregnancies. Therefore, we examined whether obese hypertensive pregnant rats have reduced PlGF and whether increasing its levels by administering recombinant human (rh)PlGF reduces their blood pressure. We utilized a genetic model of obesity characterized to be heavier, hypertensive and fertile, namely rats having heterozygous deficiency of the melanocortin-4 receptor (MC4R-def). MC4R-def obese rats had lower circulating levels of PlGF than wild-type lean controls at gestational day 19. Also, assessment of the PlGF receptor, Flt-1, in the vasculature showed that its levels were reduced in aorta and kidney glomeruli but increased in small mesenteric arteries. Chronic intraperitoneal administration of rhPlGF from gestational day 13-19 significantly increased circulating PlGF levels in both obese and lean rats, but reduced blood pressure only in the obese pregnant group. The rhPlGF treatment did not alter maternal body and fat masses or circulating levels of the adipokines, leptin and adiponectin. In addition, this treatment did not impact average foetal weights but increased placental weights regardless of obese or lean pregnancy. PlGF is reduced in MC4R-def obese hypertensive pregnant rats, which is similar to findings in obese hypertensive pregnant women, while increasing its levels with exogenous rhPlGF reduces their blood pressure.

Effect of Placental Growth Factor on Trophoblast-Endothelial Cell Interactions In Vitro.

Placental growth factor (PlGF) is an important angiogenic factor which has an emerging role in the clinical management of suspected preeclampsia. The role of PlGF in normal placental development is not completely understood and it is uncertain whether PlGF influences trophoblast and endothelial cell interactions central to uterine spiral artery remodelling, especially in variable oxygen conditions. A two-cell model of endovascular invasion was used. Tissue culture plates were coated with Matrigel™, on which fluorescent-labelled uterine microvascular endothelial cells (1 × 105/well) and HTR8/SVNeo cells were co-cultured (1 × 105/well) for 20h. Co-cultures were treated with recombinant human PlGF (rhPlGF) (10 or 100ng/mL) and incubated at either 21% O2 or 2% O2. Images were captured by fluorescence microscopy and analysed using ImageJ (n = 7). Data was analysed using SPSSv24. Treatment with rhPlGF did not improve integration in co-cultures irrespective of oxygen conditions but increased proliferation in 2% O2 of both trophoblast and endothelial cells. Expression of angiogenic factors VEGF, sFLT-1, PlGF and CXCL12 in both co-cultures and in isolated trophoblast cells was not altered by rhPlGF treatment. Expression of TLR-3 mRNA in co-cultures was increased by rhPlGF 100ng/mL at 21% O2 (p = 0.03). PlGF contributes to trophoblast and endothelial cell proliferation in the setting of physiological hypoxia but does not influence trophoblast and endothelial cell interactions in an in vitro model of spiral artery remodelling. Upregulation of TLR-3 expression in co-cultures may indicate a role for PlGF in the placental inflammatory response.

Effect of placental growth factor in models of experimental pre-eclampsia and trophoblast invasion.

Placental growth factor (PlGF) is decreased in early gestation of pregnant women who subsequently develop pre-eclampsia. In this study, pre-emptive treatment with PlGF to prevent pre-eclampsia was evaluated in an in vivo rodent model of experimental pre-eclampsia (EPE) induced by TNF-α and in an in vitro model of human first-trimester trophoblast invasion. Pregnant C57/BL6 mice were treated with recombinant mouse placental growth factor-2 (rmPlGF-2) 100μg/kg/day IP from gestational day (gd) 10. Animals had EPE induced by continuous TNF-α infusion on gd 13 and were subject to either continuous blood pressure monitoring by radiotelemetry throughout pregnancy or live placenta T2 -weighted magnetic resonance imaging (MRI) to demonstrate placental function on gd 17. There was no difference in BP (P>.99), proteinuria (P=.9) or T2 values on MRI (P=.9) between control and rmPlGF-2-treated animals. On gd 13, animals treated with rmPlGF-2 demonstrated increased placenta PlGF (P=.01) and Toll-like receptor-3 (P=.03) mRNA expression as compared with controls. Fluorescent-labelled human uterine microvascular endothelial cells and HTR8/SVNeo cells were co-cultured on Matrigel™ and treated with recombinant human PlGF (rhPlGF) (10ng/mL) and/or TNF-α (0.5ng/mL). Trophoblast integration into endothelial networks was reduced by added TNF-α (P=.006), as was rhPlGF concentration in conditioned media (P<.0001). Cell integration was not ameliorated by addition of rhPlGF (P>.9). Although TNF-α-induced EPE was not reversed with pre-emptive rmPlGF-2, a further trial of pre-emptive rhPlGF in vivo is required to determine whether the absence of effect of rhPlGF demonstrated in vitro precludes PlGF as a preventative therapy for pre-eclampsia.

Sustained Placental Growth Factor-2 Treatment Does Not Aggravate Advanced Atherosclerosis in Ischemic Cardiomyopathy.

Angiogenic growth factor therapy for ischemic cardiovascular disease carries a risk of stimulating atherosclerotic plaque growth. We evaluated risk benefit ratio of sustained administration of recombinant human placental growth factor (rhPlGF)-2 in mice with advanced atherosclerosis and chronic ischemic cardiomyopathy. We maintained apolipoprotein E-deficient mice on a high cholesterol diet and induced myocardial infarction by transient ligation at 4weeks. At 8weeks, we assessed left ventricular (LV) function and randomized mice to receive rhPlGF-2 or vehicle (VEH) subcutaneously for 28days. Administration of rhPlGF-2 significantly increased PlGF plasma levels without adverse hemodynamic or systemic inflammatory effects. RhPlGF-2 did not increase plaque area, composition, or vulnerability in the aortic arch. RhPlGF-2 significantly improved contractile function and reduced LV end-systolic and end-diastolic volume indices with a concomitant increase in capillary and arteriolar density in ischemic myocardium. RhPlGF-2 may represent a promising therapeutic strategy in chronic ischemic cardiomyopathy.

Placental Growth Factor Reduces Blood Pressure in a Uteroplacental Ischemia Model of Preeclampsia in Nonhuman Primates.

An imbalance in the angiogenesis axis during pregnancy manifests as clinical preeclampsia because of endothelial dysfunction. Circulating soluble fms-like tyrosine kinase 1 (sFLT-1) increases and placental growth factor (PlGF) reduces before and during disease. We investigated the clinical and biochemical effects of replenishing the reduced circulating PlGF with recombinant human PlGF (rhPlGF) and thus restoring the angiogenic balance. Hypertensive proteinuria was induced in a nonhuman primate (Papio hamadryas) by uterine artery ligation at 136 days gestation (of a 182-day pregnancy). Two weeks after uteroplacental ischemia, rhPlGF (rhPlGF, n=3) or normal saline (control, n=4) was administered by subcutaneous injection (100 μg/kg per day) for 5 days. Blood pressure was monitored by intra-arterial radiotelemetry and sFLT-1 and PlGF by ELISA. Uteroplacental ischemia resulted in experimental preeclampsia evidenced by increased blood pressure, proteinuria, and endotheliosis on renal biopsy and elevated sFLT-1. PlGF significantly reduced after uteroplacental ischemia. rhPlGF reduced systolic blood pressure in the treated group (-5.2±0.8 mm Hg; from 132.6±6.6 mm Hg to 124.1±7.6 mm Hg) compared with an increase in systolic blood pressure in controls (6.5±3 mm Hg; from 131.3±1.5 mm Hg to 138.6±1.5 mm Hg). Proteinuria reduced in the treated group (-72.7±55.7 mg/mmol) but increased in the control group. Circulating levels of total sFLT-1 were not affected by the administration of PlGF; however, a reduction in placental sFLT-1 mRNA expression was demonstrated. There was no significant difference between the weights or lengths of the neonates in the rhPlGF or control group; however, this study was not designed to assess fetal safety or outcomes. Increasing circulating PlGF by the administration of rhPlGF improves clinical parameters in a primate animal model of experimental preeclampsia.

Clinical Implications

Clinical Implications

Abstract 16121: Recombinant Human Placental Growth Factor 2 is a Safe and Effective Therapy for Ischemic Cardiomyopathy in Atherosclerotic Mice

Aim: Angiogenic growth factor therapy carries a risk of stimulating atherosclerotic plaque growth. We evaluated whether systemic infusion of recombinant human placental growth factor (rhPlGF) 2 improves myocardial neovascularization, left ventricular (LV) function and adverse remodeling in a murine model of advanced atherosclerosis and chronic myocardial infarction (MI) without increasing atherosclerotic plaque size and plaque vulnerability. Methods: ApoE -/- mice were fed a high cholesterol diet and MI was induced 4 weeks (w) later using 60 min LAD occlusion followed by reperfusion. After 8 w, we assessed LV function using echocardiography and randomized mice to receive rhPlGF2 (450μg/kg/day, n=20) or PBS (n=20) via osmotic minipumps for 28 days. Echocardiography and histological analyses were performed at 12 and 20 w. Results: Infusion of rhPlGF2 increased PlGF plasma levels for 3 w up to ~1600-fold without adverse side effects, or changes in total cholesterol and high sensitive CRP levels. In rhPlGF2-treated mice, capillary and arteriolar density was significantly higher in ischemic myocardium (2813±212 capillaries/mm 2 at 12 w vs 2144±478 in PBS, P &lt;0.05; 125±18 arterioles/mm 2 at 20 w vs 77±13 in PBS, P =0.001). RhPlGF2 significantly improved ejection fraction (EF), reduced LV end-systolic and end-diastolic volume indices at 12 w and prevented further LV dilation and EF deterioration at 20 w (Figure). RhPlGF2 did not increase plaque area in the aortic arch, or the degree of fibrosis, calcification, capillary or arteriolar density and MAC3-positive cell areas in plaques at 12 and 20 w. Conclusion: Systemic rhPlGF2 infusion significantly improves neovascularization and contractile function, and prevents LV adverse remodeling in chronic ischemic cardiomyopathy without increasing atherosclerotic plaque burden or plaque vulnerability. RhPlGF2 may represent a promising and safe therapeutic strategy in chronic ischemic cardiomyopathy.

An unexpected tail of VEGF and PlGF in pre-eclampsia

PET (pre-eclamptic toxaemia), characterized by pregnancy-related hypertension and proteinuria, due to widespread endothelial dysfunction, is a primary cause of maternal morbidity. Altered circulating factors, particularly the VEGF (vascular endothelial growth factor) family of proteins and their receptors, are thought to be key contributors to this disease. Plasma from patients with PET induces numerous cellular and physiological changes in endothelial cells, indicating the presence of a circulating imbalance of the normal plasma constituents. These have been narrowed down to macromolecules of the VEGF family of proteins and receptors. It has been shown that responses of endothelial cells in intact vessels to plasma from patients with pre-eclampsia is VEGF-dependent. It has recently been shown that this may be specific to the VEGF₁₆₅b isoform, and blocked by addition of recombinant human PlGF (placental growth factor). Taken together with results that show that sVEGFR1 (soluble VEGF receptor 1) levels are insufficient to bind VEGF-A in human plasma from patients with pre-eclampsia, and that other circulating macromolecules bind, but do not inactivate, VEGF-A, this suggests that novel hypotheses involving altered bioavailability of VEGF isoforms resulting from reduced or bound PlGF, or increased sVEGFR1 increasing biological activity of circulating plasma, could be tested. This suggests that knowing how to alter the balance of VEGF family members could prevent endothelial activation, and potentially some symptoms, of pre-eclampsia.

Abstract 2453: P16, a novel monoclonal antibody specifically recognizing human placental growth factor (PLGF), blocks PLGF-VEGFR1 interaction in vitro and inhibits angiogenesis and tumor growth in vivo

Abstract Placental growth factor play (PLGF) is a homolog of vascular endothelial growth factor (VEGF) and binds to VEGF receptor-1. Previous studies have indicated its synergistic role with VEGF in angiogenesis, while a recent study suggested its independent role in promoting growth and metastasis of VEGF-inhibitor-resistant tumors. In order to further investigate the role of PLGF in tumor angiogenesis in relationship to VEGF, we have generated a novel PLGF-specific monoclonal antibody (mAb). Hybridomas secreting PLGF-specific mAbs were obtained by fusing splenocytes from mice immunized with yeast-derived recombinant human PLGF protein with Sp/20 myeloma cells; the mAb clone that showed highest binding affinity, designed as P16, was selected for further study. This P16 mAb recognizes human PLGF, in both native and denatured forms, but not VEGF-A, VEGF-B or VEGF-D. P16 mAb is of IgG1-kappa type. Sequence analysis of cDNA encoding the heavy and light chains of P16 mAb showed novel variable regions. Using P16 mAb in immunohistochemical staining assay, increased levels of PLGF protein were found in a number of human tumors from lung, breast and colon. PLGF proteins were also detected in a wide variety of normal human tissues including lung, stomach, colon and kidney, contrary to previous reports. Importantly, P16 mAb blocked the binding of PLGF to VEGFR-1 in a dose-response manner, and inhibited the PLGF-mediated endothelial cell line growth in vitro. Treatment of human tumor xenografts (A673 rhabdomyosarcoma) in nude mice with P16 mAb resulted in marked inhibition of tumor growth, which is similar in degree to the tumor inhibition by VEGF-specific antibody Bevacizumab. A humanized form of P16 mAb was constructed and showed similar inhibition of tumor growth in xenograft nude mice model. In conclusion, P16 mAb is a novel mAb specifically recognizing human PLGF but not VEGF and blocks the binding of PLGF to its receptor. It inhibits angiogenesis and tumor growth, suggesting an important and independent role of PLGF in tumor growth. Further studies on its mechanisms of action and its clinical applications are under way. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2453.

Abstract 1388: sFLT01: An anti-angiogenic fusion protein that neutralizes placental growth factor (PlGF) and vascular endothelial growth factor (VEGF)

Abstract Introduction: PlGF and VEGF stimulate angiogenesis and promote the growth of tumor vasculature. PlGF is a member of the VEGF family and binds to VEGFR1. sFLT01 is a novel fusion protein comprised of the Fc portion of human IgG1 and the PlGF- and VEGF-binding domain of VEGFR1/Flt-1. The properties of sFLT01 and the potential of sFLT01 as an anti-angiogenic agent to inhibit tumor growth were investigated in several in vitro assays and in multiple xenograft tumor models. Methods: The binding kinetics of sFLT01 for both the human and murine homologues of PlGF and VEGF were assessed by Biacore. The abilities of recombinant human PlGF and VEGF to induce endothelial cell and pericyte proliferation and of sFLT01 to inhibit this stimulation were investigated in cell-based assays. The secretion of human VEGF and PlGF in culture by the HT29 colon carcinoma, H460 lung carcinoma, and A673 sarcoma human cell lines was quantified by ELISA. In efficacy studies, sFLT01 was administered by intraperitoneal injection twice per week to immunodeficient mice bearing HT29, H460, or A673 subcutaneous tumors. Antibodies specific for human IgG and VEGR2 were applied to A673 sarcoma tumor sections from mice treated with sFLT01 to visualize sFLT01 in the tumors and determine VEGFR2 expression in the cellular components. Pericytes and endothelial cells were identified with antibodies against NG2 and CD31. Results: The Biacore results indicated that sFLT01 has high affinity for human and murine PlGF and VEGF. Human recombinant PlGF and VEGF each induced the proliferation of human pericytes and endothelial cells in culture. This stimulation was inhibited by sFLT01. A673 sarcoma, HT29 colon and H460 lung carcinoma cells secreted higher levels of VEGF than PlGF in culture. In vivo, 10 mg/kg sFLT01 was effective at significantly slowing the growth of HT29 colon carcinoma and A673 sarcoma tumors compared to controls. Further analysis of the A673 sarcoma tumors in sFLT01-treated mice by immunohistochemistry revealed that sFLT01 penetrated multiple areas of the tumor. sFLT01 was detected in the vasculature, stroma, necrotic areas, and adjacent to malignant cells. sFLT01 treatment in the A673 model disrupted vessel integrity with a lack of association between endothelial cells and pericytes. A673 sarcoma cells expressed VEGFR2 in vivo. Conclusion: sFLT01 neutralizes the angiogenic activity of multiple vasculogenic VEGF family members in vitro and inhibited the proliferation of cells that form blood vessels, endothelial cells and pericytes. In vivo, sFLT01 treatment resulted in disorganized tumor vasculature thereby slowing the growth of xenografts tumors. The expression of VEGFR2 in A673 sarcoma tumors suggests that VEGF may play a role in autocrine signaling in some malignant cells. sFLT01 has antitumor and antiangiogenic activity in several human tumor xenografts and may offer therapeutic benefit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1388.

Serum midkine predicts cardiac events in patients with heart failure

Background: Recent our clinical work demonstrated that placental growth factor (PlGF), a member of VEGF family, is rapidly produced within infarct myocardial tissue, and the plasma level of PlGF is positively correlated with the number of monocytes and CD34-positive cells, and improvement of left ventricular function. However, therapeutic effects of PlGF onMI are not understood. Methods and results: One day before ligation of coronary artery, recombinant human PlGF (rhPlGF) (1 μg) was infused into the peritoneal cavity of C57BL/6 mice through subcutaneously implanted osmotic mini pump until 2 days after the ligation. After 28 days, survival rate was significantly higher in rhPlGF-treated group (70.0%) than PBS-treated group (33.3%) (pb0.05). Seven days after MI, the infarct area, stained by 1.5% TTC (2,3,5-triphenyltetrazolium chloride), was smaller in rhPlGF-treated group (4.25±2.04 mm) than PBS-treated group (5.95±1.54 mm), and it indicates that exogenous PlGF suppressed cardiac remodeling. Echocardiography also revealed that left ventricular diastolic diameter was decreased (rhPlGF: 4.50±0.46 mm, PBS: 5.14±0.38 mm, pb0.01), and ejection fraction was increased in rhPlGF-treated group (rhPlGF: 40.6+9.17%, PBS: 25.7+7.23%, pb0.01). Histological analysis showed that both CD31-positive vascular endothelial cells (rhPlGF: 644.6+ 90.54/mm, PBS: 459.0+ 73.89/mm, pb0.01) and α-smooth muscle actin-positive vessels (rhPlGF: 31.6+7.20/mm, PBS: 23.5+7.41/mm, pb0.01) was increased at infarct area. Conclusion: rhPlGF treatment preserved left ventricular function and inhibited post-infarct remodeling by enhancing neovascularization and arteriogenesis, resulting in improvement of survival rate after myocardial infarction.